Real-time two-photon microscopy and its application for in situ imaging

The recent developments in fluorescent-probes and laser technologies have provided us opportunities to observe the biological functions in living organs and tissues under a microscope. Although single-photon excitation confocal microscopy is an indispensable tool for observing these specimens, the observable depth of this microscopy is still not enough to analyze the functions of whole parts of organs in living organisms. To observe the deeper parts, we have attempted to develop new real-time two-photon excitation microscopy equipped with a microlens-arrayed multipinhole scanning device. Using this real-time two-photon microscope, we examined the dynamics of the intracellular calcium concentration ([Ca2+](i)) in the cardiac myocytes in the rat whole heart to understand their physiological functions and mechanisms. We could observe Ca2+ waves on the surface in the whole heart. However, we still need higher-energy laser power for two-photon excitation and visualization of [Ca2+](i) dynamics in the deeper parts of the whole heart.